1. Field of the Invention
The invention relates to microspheres and delivery of therapeutic agents, and more specifically, to microspheres coated with surface-layer proteins having affinity for certain tissues for delivery of a therapeutic agent, such as, delivery of defensins, particularly HD5α in the treatment of inflammatory conditions of the bowel, such as Crohn's disease and/or ulcerative colitis.
2. Description of the Related Art
The surface properties and surface structures of bacteria are important in regulating bacterial adhesion to host cells [1]. Adherence to constituents of the extracellular matrix, such as glycoproteins like fibronectin and laminin as well as to different types of collagen, has been shown to be mediated through surface layer proteins in various bacterial species [2, 3]. These adhesive properties have been linked to the ability of bacteria to express virulence [4-7] as well as probiotic properties [8, 9].
In the case of Lactobacilli, various studies have shown the importance of the surface-layer or S-layer protein in adhesion of the bacterium to the intestinal epithelium [9-11]. S-layers are one of the most common surface structures of many archaebacteria and eubacteria. A shared feature of S-layers is the ability to self-assemble and form a regularly ordered, planar array of proteinaceous subunits [12]. Various functions have been assigned to S-layers. Most species of lactobacilli contain two or more genes encoding S-layer proteins. Three slp genes (slpB, slpC and slpD) have been identified in L. brevis ATCC 14869 whereas only a single gene slpA has been identified L. brevis ATCC 8287. Because S-layer lattices show identical pore size and morphology, they act as precise molecular sieves for the cell. They act as a protective shield against exceptionally high ion concentrations as well as certain bacterial parasites [13]. The SlpA surface protein of Lactobacillus brevis is reported to be essential for adhesion of this bacterium to intestinal cells through its interaction with the extracellular matrix component fibronectin [11].
Microbial infection and inflammation is a constant risk in the mucosal surface of the gastrointestinal tract, which comprises the first line of defense against a variety of microorganisms. Remarkably, this mucosal immune system has the ability to distinguish pathogens from the commensal microflora and to elicit the appropriate immune response [14-16]. The intestinal mucosa is covered by a single layer of epithelial cells; separating mucosa associated lymphoid tissue from surface antigens. The epithelium provides both a barrier as well as a signaling function against infection [17]. The epithelial cells as well as immune cells of the subepithelial compartment, including lymphocytes, monocytes, macrophages, polymorphonuclear leukocytes and dendritic cells, induce the host innate and adaptive immune systems. Activation of the innate host defense by microbes occurs via the specific recognition of microbial molecules, known as pathogen-associated molecular patterns or PAMPs [18].
Host cells detect these patterns through receptors, including members of the Toll-like receptor family and the nucleotide-binding site/leucine rich repeat proteins such as NOD1 and NOD2 [19-21]. Toll-like receptors are type I integral membrane glycoproteins belonging to the superfamily of interleukin-1 receptors. They play an essential role in the initiation of the innate antimicrobial immune response in plants, insects and mammals [22]. The extracellular part of the receptors contains copies of a motif known as leucine-rich repeat, thought to be directly involved in the recognition of microbial components. In humans, ten Toll-like receptors can be found, designated TLR1-TLR10, each recognizing conserved microbial molecules [23-27]. Toll-like receptors are localized on the cell surface or in endosome-lysosome intracellular compartments of immune cells [28,29]. Most of the current studies on the biological role and distribution of Toll-like receptors have focused on dendritic cells and macrophages. In contrast, the expression patterns and role of TLR's in antigen presenting cells present in the mucosal surface as well as epithelial cells has been poorly described.
Crohn's disease is a chronic disease of the intestine characterized by inflammation of the gut. The disease can be located from the oral cavity to the rectum although in almost 70% of patients the distal ileum is affected. Crohn's disease and ulcerative colitis constitute the two major chronic inflammatory bowel diseases (IBD's), affecting one in 500 individuals [30]. The etiopathogenesis of Crohn's disease has been linked recently to a diminished expression of intestinal anti-microbial peptides called defensins [31-36].
Defensins are cationic and cysteine-rich peptides with molecular weights of 3 to 5 kDa. Based on the connectivity of the six cysteine residues, human defensins are classified into α and β subfamilies [37-39]. The α-defensins are predominantly expressed in neutrophils (human neutrophil peptide 1-4) or specialized cells of the intestinal epithelium called Paneth cells (human defensin 5 and 6α) [40,41]. Human β-defensins are found predominantly in various epithelial cells and tissues. Defensins play an important role in the host innate immune defense, not only showing antibiotic and antifungal activity but also kill certain enveloped viruses and act selectively cytotoxic to tumor cells [42,43]. Importantly, human α and β-defensin also act as immune modulators in adaptive immunity [44].
Initial studies showed that α-defensins chemoattract monocytes [45]. Subsequently, α-defensins were reported to chemoattract different subsets of T lymphocytes and immature dendritic cells [46,47]. More recently, similar functions have been reported for β-defensins [48-50]. These studies showed that β-defensins selectively chemoattract memory T cells and immature dendritic cells. In addition, it was shown that human β-defensin 2 act directly as endogenous ligands for Toll-like receptors, mediating signaling for dendritic cell maturation in vivo. Defensins and chemokines share a striking structural resemblance, adopting a disulfide stabilized core structure of three anti-parallel β-sheets [51].
The recent discovery of several genetic loci showing a significant association with Crohn's disease has greatly enhanced the understanding of underlying pathogenic mechanisms. In particular, NOD2 has been identified as the first gene firmly associated with Crohn's disease susceptibility [52,53]. NOD2 is a cytoplasmic protein that senses components of the microbial cell wall and regulates inflammatory processes and apoptosis. The protein is composed of two N-terminal caspase recruitment domains, a nucleotide binding and oligomerization domain and contains ten leucine-rich repeats at its C-terminus. It is expressed constitutively in particular in macrophages, neutrophils and dendritic cells [54] as well as Paneth cells in the small intestine [55]. NOD2 specifically recognizes muramyl dipeptide, a peptidoglycan motif present in the cell wall of both gram-negative and gram-positive bacteria [56]. After recognition, NOD2 has been shown to activate signaling pathways involved in inflammation in vitro through NF-κB activation [57,58]. In addition, NOD2 activation has been linked to innate immunity [59]. In NOD2 knockout mice, expression of α-defensins was markedly decreased suggesting that NOD2 plays a role in intestinal host defense. This provides further evidence to the notion that defensins and their relation to Toll-like receptors may play a key role in maintaining immune homeostasis.
Importantly, human α and β-defensin also act as immune modulators in adaptive immunity [44]. Further, the etiopathogenesis of Crohn's disease has been linked recently to a diminished expression of the anti-microbial peptides defensins [31-36]. Thus, it would be advantageous to provide a method for delivery of defensins to intestinal tissue to provide an increase of this intestinal anti-microbial peptide defensins.